CN113717184A

CN113717184A - Quinoline alkaloid with tobacco mosaic virus resisting activity in cigar, and preparation method and application thereof

Info

Publication number: CN113717184A
Application number: CN202110953875.3A
Authority: CN
Inventors: 吴玉萍; 孔光辉; 蔺忠龙; 赵高坤; 姚恒; 张光海; 李薇; 阴根云; 肖东; 师建全; 杨光宇; 胡秋芬
Original assignee: China Tobacco Yunnan Industrial Co Ltd; Yunnan Academy of Tobacco Agricultural Sciences
Current assignee: China Tobacco Yunnan Industrial Co Ltd; Yunnan Academy of Tobacco Agricultural Sciences
Priority date: 2021-08-19
Filing date: 2021-08-19
Publication date: 2021-11-30
Anticipated expiration: 2041-08-19
Also published as: CN113717184B

Abstract

The invention discloses quinoline alkaloid (I) and a preparation method and application thereof. Extracting cigar tobacco leaf with high concentration methanol, high concentration acetone/water or high concentration ethanol/water, mixing extractive solutions, filtering, and concentrating under reduced pressure to obtain extract; packing the extract into a column by a silica gel dry method for silica gel column chromatography; performing gradient elution by using chloroform-acetone solution; and further separating and purifying the 7:3 part of the eluent by using high pressure liquid chromatography to obtain the required quinoline alkaloid. The invention also discloses the application of the compound, and activity tests show that the compound has good inhibition effect on tobacco mosaic virus. The compound has novel structure and better anti-virus activity, and can be used as a lead compound for resisting tobacco mosaic virus for research and development of a tobacco mosaic virus-resisting pharmaceutical preparation.

Description

Quinoline alkaloid with tobacco mosaic virus resisting activity in cigar, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of comprehensive utilization of cigars, and particularly relates to quinoline alkaloid extracted from cigars for the first time. Meanwhile, the invention also relates to a preparation method of the compound and application of the compound in resisting tobacco mosaic virus.

Background

Tobacco is the most complex plant in the world in terms of chemical composition, and secondary metabolites are very abundant, and as reported by Dube and Green et al in 1982, the chemical compositions identified in tobacco are more than 2549, and by 2008, the total number of compounds found in tobacco, tobacco substitutes, and cigarette smoke is about 8700, as reported by Rodgman and perfetti. At present, more than 3000 monomer chemical substances are identified from tobacco, and many ingredients are not identified yet.

Cigars belong to the tobacco family of the Nicotiana species, the variety Nicotiana tabacum. After tobacco planting, airing and fermenting, the cigar is rolled into a finished product for consumers to suck. Cigars are an important component of Chinese tobacco core competitiveness and are a beneficial complement to cigarette consumption. Since 2012, the domestic cigar market has been explosively increased, the annual average sales increase is kept above 40%, and by 2019, the domestic manual cigar production and sales volume has broken through 540 thousands of cigars. The cigar market in China has been on an initial scale, and domestic cigar consumer groups are continuously developing and growing. In addition, the cigar is mainly used for cigarette smoking, and various chemical components with useful values can be extracted from the cigar, so that a new guiding compound is provided for the development of medicaments and biological pesticides.

Quinoline alkaloid compounds exist in a plurality of natural plants, and the alkaloid has extremely strong physiological activity and is a hot spot for research, development and utilization at home and abroad. Whether extracted from natural plants or artificially synthesized or modified molecular structures and the research of structure-activity relationship, isoquinoline alkaloids are always the source for searching lead compounds and bioactive components with development and application prospects. In order to find more active compounds from the plants in the genus Nicotiana, develop and process the non-smoking application of the tobacco, the invention separates and obtains a new quinoline alkaloid from Yunnan cigar tobacco leaves, the compound has not been reported so far, and the compound is worth mentioning that the compound has obvious anti-tobacco mosaic virus activity.

Disclosure of Invention

The invention aims to provide a novel quinoline alkaloid.

It is another object of the present invention to provide a method for preparing said quinoline alkaloid.

The invention also aims to provide application of the quinoline alkaloid in resisting tobacco mosaic virus.

All percentages used in the present invention are mass percentages unless otherwise indicated.

The invention separates a new quinoline alkaloid from cigar, the molecular formula of which is C₁₃H₁₁NO₃Having the following structural formula:

the compound was named: (7-methoxy-furan [3,2-c ] quinolin-6-yl) -methanol, english name: (7-methoxy-furo [3,2-c ] quinolin-6-yl) -methanol as a pale yellow gum.

A process for preparing said quinoline alkaloid, the process comprising the steps of:

(1) extracting the extractum: crushing a tobacco sample, using high-concentration methanol (w%: 80-100%) or high-concentration ethanol (w%: 80-100%) or high-concentration acetone (w%: 60-90%) as an extraction solvent: soaking cigars for 24-72 h at a weight ratio of 2-4: 1, extracting for 3-5 times, combining the extracting solutions, filtering and concentrating into an extract;

(2) silica gel column chromatography: dissolving the extract with 1.5-3 times of pure methanol or pure ethanol or pure acetone, mixing with 0.8-1.2 times of 80-100 mesh silica gel, and performing silica gel column chromatography with 2-4 times of 160-300 mesh silica gel in a dry method; gradient eluting with chloroform-acetone solution at volume ratio of (1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1 and 1:2), mixing the same fractions, collecting eluate of each fraction, and concentrating;

(3) high-pressure liquid chromatography separation and purification: and (3) further separating and purifying the 7:3 part of the column chromatography eluent by using high pressure liquid chromatography to obtain the quinoline alkaloid.

The high pressure liquid chromatography is used for separating and purifying C with the size of 21.2mm multiplied by 250mm and the diameter of 5 mu m₁₈And (3) carrying out chromatographic column chromatography, wherein the flow rate is 20mL/min, the mobile phase is 52% methanol, the detection wavelength of an ultraviolet detector is 334nm, 200 mu L of sample is injected each time, a chromatographic peak of 30.2min is collected, and the chromatographic peak is evaporated to dryness after multiple accumulation.

After the separation and purification by the high performance liquid chromatography, a preferable subsequent treatment scheme is that the obtained compound is dissolved by pure methanol again, and then the obtained compound is separated by gel column chromatography by taking the pure methanol as a mobile phase so as to be further separated and purified.

TABLE-1 preparation of the compounds¹H NMR and¹³c NMR data (CDCl)₃)

The structure of the quinoline alkaloid prepared in the above manner was determined by the following method. The compounds of the present invention are pale yellow gels. High resolution mass spectrometry (HR-ESIMS) gives the excimer ion peak M/z 252.0645[ M + Na [ ]]⁺(calculated 252.0637). Bonding of¹H and¹³the C NMR spectrum gives a molecular formula C₁₃H₁₁NO₃The unsaturation degree was 9. Its infrared spectrum shows hydroxyl (3389 cm)^-1) Carbon to nitrogen bond (1819 cm)^-1) And aromatic rings (1614,1469,1374 cm)^-1) The structure and the maximum absorption of ultraviolet spectrum at 210, 242 and 334nm prove that aromatic ring conjugated structures exist in the compound. Process for preparing compounds¹H and¹³c NMR and DEPT data (Table-1) show the presence of 13 carbons and 11 hydrogens in the compound, containing 1,2,4, 5-tetrasubstituted benzene ring (C-5 to C-8, C-4a and C-8 a; H-5 and H-8), one furan moiety (C-2 and C-3, H-2 and H-3), one hydroxymethyl group (-CH)₂-OH, C-1 'and H-1'), one methoxy group (-MeO-7, delta)_H3.78s and delta_C56.3q), and one-C-CH-N-structural fragment (C-4, C-3a, C-9 a; h-9 a).

According to its nuclear magnetic resonance signal, the benzene ring, the furan ring and the-C-CH-N-structural fragment contain 8 of 9 unsaturations, 1 quinoline ring should be formed between the benzene ring and the-C-CH-N-structural fragment to satisfy the 9 unsaturations, and it is presumed that the compound may be a furan [3,2-C ] quinoline structure. This speculation is further confirmed by HMBC correlation of H-5 and C-4, C-4a, C-8a, H-8 and C-4a, C-8a, H-9a and C-3, C-4, C-3a, C-8a, H-2 and C-4, C-3a, and H-3 and C-4, C-3a, C-9 a.

Furan [3,2-c ] in compounds]After the quinoline backbone is confirmed, the positions of the other substituents (hydroxymethyl and methoxy) can also be confirmed by analyzing their HMBC associations. The substitution of the hydroxymethyl group at the C-6 position can be determined by correlation of H-1' with HMBC at C-5, C-6, C-7, and further by methoxyhydrogen (. delta.) (_H3.78s) and HMBC correlation at C-7 determined that the methoxy substitution was at the C-7 position. To this end, the compoundsThe structure of (a) was confirmed and named: (7-methoxy-furan [3, 2-c)]Quinolin-6-yl) -methanol.

Infrared, ultraviolet and mass spectral data of compounds: UV (MeOH), λ_max(logε)210(4.58)、242(4.32)、334(3.90)nm；IR(KBr)ν_max 3389、3084、2322、1819、1614、1469、1374、162、1058、936cm^-1(ii) a ESI-MS (Positive ion mode) M/z 252[ M + Na ]]⁺(ii) a HR-ESI-MS (positive ion mode) M/z 252.0645[ M + Na ]]⁺(calculated 252.0637, C₁₃H₁₁NNaO₃)。

The results of the activity test of the compound of the invention against tobacco mosaic virus by using a semi-leaf method show that the relative inhibition rate of the compound is 42.2 percent and exceeds the relative inhibition rate of the control ningnanmycin by 32.5 percent, which shows that the compound has good activity against tobacco mosaic virus.

The compound of the invention is separated for the first time, is determined as quinoline alkaloid by the nuclear magnetic resonance and mass spectrometry determination method, and characterizes the specific structure. The activity detection result shows that the compound has good application prospect in preparing anti-rotavirus medicaments. The compound has simple structure and good activity, is easy to extract and separate and realize subsequent artificial synthesis, can be used as a leading compound for research and development of anti-mosaic virus medicaments, and is used for research and development of anti-mosaic virus medicinal preparations.

Drawings

FIG. 1 is a nuclear magnetic resonance carbon spectrum of a quinoline alkaloid of the present invention;

FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of a quinoline alkaloid of the present invention;

FIG. 3 is a graph of the major HMBC correlation of the quinoline alkaloids of the present invention.

Detailed Description

The invention is further described in detail below with reference to the drawings and examples, but the invention is not limited in any way, and any changes or modifications made based on the teachings of the invention fall within the scope of the invention.

Example 1

Preparation of quinoline alkaloid C₁₃H₁₁NO₃The method comprises the steps of extract extraction, silica gel column chromatography and high-pressure liquid chromatography separation, and specifically comprises the following steps:

1. extracting the extractum: crushing tobacco leaves, and extracting with high-concentration methanol (w%: 95%) or high-concentration ethanol (w%: 95%) or high-concentration acetone (w%: 70%) as an extraction solvent: soaking cigar (weight ratio) 3:5 for 54h, extracting for 4 times, mixing extractive solutions, filtering, and concentrating into extract.

2. Silica gel column chromatography: dissolving the extract with 2.8 times of pure methanol or pure ethanol or pure acetone, mixing with 1.2 times of 80-100 mesh silica gel, and performing silica gel column chromatography with 3 times of 250 mesh silica gel by dry method; gradient eluting with chloroform-acetone solution at volume ratio of (1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1 and 1:2), mixing the same fractions, collecting eluate of each fraction, and concentrating.

3. High-pressure liquid chromatography separation: separating and purifying 7:3 part of the column chromatography eluent by high pressure liquid chromatography to obtain the quinoline alkaloid, wherein the high pressure liquid chromatography separation and purification adopts C of 21.2mm × 250mm and 5 μm₁₈And (3) carrying out chromatographic column chromatography, wherein the flow rate is 20mL/min, the mobile phase is 52% methanol, the detection wavelength of an ultraviolet detector is 334nm, 200 mu L of sample is injected each time, a chromatographic peak of 30.2min is collected, and the chromatographic peak is evaporated to dryness after multiple accumulation.

Separating the purified substance by high pressure liquid chromatography, and preferably performing a post-treatment by dissolving the obtained compound again with pure methanol, and separating with gel column chromatography with pure methanol as mobile phase for further separation and purification.

The tobacco raw materials used in the invention are not limited by regions and varieties, and the invention can be realized, and the invention is further explained by the tobacco raw materials from different production places of tobacco industry Limited liability companies in Yunnan, China, in the following:

example 2

The cigar sample is from Yuxi Yunan, and the variety is Yuxue No. 8. Sampling cigar 2.0kg, pulverizing, extracting with 95% methanol for 5 times, each time for 24 hr, mixing extractive solutions, filtering, and concentrating under reduced pressure to obtain extract 118 g. Dissolving the extract with 2.0 times of pure methanol by weight, mixing with 136g of 100 mesh crude silica gel, loading 0.4kg of 160 mesh silica gel into a column, performing silica gel column chromatography, performing gradient elution with chloroform-acetone in volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1 and 1:2, monitoring by TLC, and combining the same parts to obtain 8 parts, wherein the chloroform-acetone eluted part in volume ratio of 7:3 is separated by using an Agilent 1100 semi-preparative high performance liquid chromatography, 52% methanol is used as a mobile phase, a Zorbax SB-C18(21.2 × 250mm,5 μm) preparation column is used as a stationary phase, the flow rate is 20ml/min, the detection wavelength of an ultraviolet detector is 334nm, sampling 200 μ L each time, collecting 30.2min chromatographic peaks, and evaporating to dryness after multiple accumulation; dissolving the obtained product with pure methanol again, separating with Sephadex LH-20 gel column chromatography with pure methanol as mobile phase to obtain the new compound (68.4 mg).

Example 3

Cigar sample is from Yunnan Dehong, and the variety is Yunxue No. 2, 3.5kg of cigar sample is cut into pieces, extracted with 95% ethanol for 4 times, each time for 48h, the extracting solutions are combined, filtered, and concentrated under reduced pressure to obtain extract 280 g. Dissolving the extract with 2.0 times of pure methanol by weight, mixing with 250g of 80-mesh crude silica gel, loading 1.0kg of 200-mesh silica gel into a column, performing silica gel column chromatography, performing gradient elution with chloroform-acetone in volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1 and 1:2, monitoring by TLC, and combining the same parts to obtain 8 parts, wherein the chloroform-acetone eluted part in volume ratio of 8:2 is separated by using an Agilent 1100 semi-preparative high performance liquid chromatography, 52% methanol is used as a mobile phase, a Zorbax SB-C18(21.2 × 250mm,5 μm) preparation column is used as a stationary phase, the flow rate is 20ml/min, the detection wavelength of an ultraviolet detector is 334nm, sampling 200 μ L each time, collecting 30.2min chromatographic peaks, and evaporating to dryness after multiple accumulation; dissolving the obtained product with pure methanol again, separating with Sephadex LH-20 gel column chromatography with pure methanol as mobile phase to obtain the new compound (115.6 mg).

Example 4

The cigar sample is from Yunnan Lincang, and is Yunxue No. 4, sampling cigar, 5kg pulverizing, extracting with 75% acetone by ultrasonic for 3 times, each time for 72 hr, mixing extractive solutions, filtering, and concentrating under reduced pressure to obtain extract 368 g. Dissolving the extract with 1.6 times of pure methanol by weight, mixing with 400g of 90 mesh crude silica gel, loading 1.4kg of 180 mesh silica gel into a column, performing silica gel column chromatography, performing gradient elution with chloroform-acetone in volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1 and 1:2, monitoring by TLC, and combining the same parts to obtain 8 parts, wherein the chloroform-acetone eluted part in volume ratio of 8:2 is separated by using an ansiram 1100 semi-preparative high performance liquid chromatography, 52% of methanol is used as a mobile phase, a Zorbax SB-C18(21.2 × 250mm,5 μm) preparation column is used as a stationary phase, the flow rate is 20ml/min, the detection wavelength of an ultraviolet detector is 334nm, sampling 200 μ L each time, collecting 30.2min chromatographic peaks, and evaporating to dryness after multiple accumulation; dissolving the obtained product with pure methanol again, separating with Sephadex LH-20 gel column chromatography with pure methanol as mobile phase to obtain the new compound (158 mg).

Example 5

Identification of the structure of the compounds

The structures of the quinoline alkaloids prepared in the above-described manner from the compounds prepared in examples 1 to 4 were determined by the following methods. The compound was a pale yellow gum. High resolution mass spectrometry (HR-ESIMS) gives the excimer ion peak M/z 252.0645[ M + Na [ ]]⁺(calculated 252.0637). Bonding of¹H and¹³the C NMR spectrum gives a molecular formula C₁₃H₁₁NO₃The unsaturation degree was 9. Its infrared spectrum shows hydroxyl (3389 cm)^-1) Carbon to nitrogen bond (1819 cm)^-1) And aromatic rings (1614,1469,1374 cm)^-1) The structure and the maximum absorption of ultraviolet spectrum at 210, 242 and 334nm prove that aromatic ring conjugated structures exist in the compound. Process for preparing compounds¹H and¹³c NMR and DEPT data (Table-1) show the presence of 13 carbons and 11 hydrogens in the compound, containing 1,2,4, 5-tetrasubstituted benzene ring (C-5 to C-8, C-4a and C-8 a; H-5 and H-8), one furan moiety (C-2 and C-3, H-2 and H-3), one hydroxymethyl group (-CH)₂-OH, C-1 'and H-1'), one methoxy group (-MeO-7, delta)_H3.78s and delta_C56.3q), and one-C-CH-N-structural fragment (C-4, C-3a, C-9 a; h-9 a).

Furan [3,2-c ] in compounds]After the quinoline backbone is confirmed, the positions of the other substituents (hydroxymethyl and methoxy) can also be confirmed by analyzing their HMBC associations. The substitution of the hydroxymethyl group at the C-6 position can be determined by correlation of H-1' with HMBC at C-5, C-6, C-7, and further by methoxyhydrogen (. delta.) (_H3.78s) and HMBC correlation at C-7 determined that the methoxy substitution was at the C-7 position. The structure of the compound was thus confirmed and named: (7-methoxy-furan [3, 2-c)]Quinolin-6-yl) -methanol.

Example 6

The compound prepared in example 3 was taken as a yellow gum. The assay procedure was the same as in example 5, and it was confirmed that the compound prepared in example 3 was the quinoline alkaloid- (7-methoxy-furan [3,2-c ] quinolin-6-yl) -methanol.

Example 7

The compound prepared in example 4 was taken as a yellow gum. The assay procedure was the same as in example 5, and it was confirmed that the compound prepared in example 4 was the above-mentioned — (7-methoxy-furan [3,2-c ] quinolin-6-yl) -methanol.

Example 8

The quinoline alkaloids prepared in examples 1-4 were tested for activity against tobacco mosaic virus as follows:

the activity of the compound of the invention against tobacco mosaic virus is measured by a half-leaf method when the mass concentration of the medicament is 50 mg/L. Selecting leaves suitable for testing (normal leaves, no disease and no insect) on plants of 5-6-year-old flue-cured tobacco, uniformly spraying fine carborundum on the leaves, and using a writing brush to apply a standby tobacco mosaic virus source (3.0 multiplied by 10)^-3) Uniformly smearing on leaf blades scattered with carborundum, immediately placing in a culture dish containing medicinal liquid for treatment for 20min after all selected leaf blades are disinfected,taking out, wiping off water drops and liquid medicine on the leaves, recovering and arranging the two half leaves in a glass jar paved with toilet paper for moisture preservation, covering a glass cover, controlling the temperature to be (23 +/-2) DEG C, placing the glass jar in a greenhouse for natural light irradiation, and observing withered spots after 2-3 days.

XI％＝(CK-T)/CK×100％

X: relative inhibition ratio (%), CK: the number of dead spots of half leaf after being soaked in clear water is one, and the number of dead spots of half leaf after being soaked in liquid medicine is one.

The result shows that the relative inhibition rate of the compound is 42.2 percent and exceeds the relative inhibition rate of the control ningnanmycin by 32.5 percent, which indicates that the compound has good activity of resisting tobacco mosaic virus. Can be used as a new lead compound for researching and developing biological pesticides for controlling tobacco mosaic disease.

Claims

1. A quinoline alkaloid having the following structural formula:

the compound was named: (7-methoxy-furan [3,2-c ] quinolin-6-yl) -methanol, english name: (7-methoxy-furo [3,2-c ] quinolin-6-yl) -methanol.

2. A process for the preparation of a quinoline alkaloid as claimed in claim 1, comprising the steps of:

(1) extract extraction, which is to take tobacco leaves as raw materials, crush or cut the tobacco leaves into small segments, and take methanol or ethanol with the weight percentage concentration of 80-100 percent or acetone with the weight percentage concentration of 60-90 percent as an extraction solvent; soaking the tobacco leaves for 24-72 h according to the weight ratio of 2-4: 1, extracting for 3-5 times, combining the extracting solutions, filtering and concentrating into an extract;

(2) silica gel column chromatography: the extractum is subjected to silica gel column chromatography by using a 160-300-mesh silica gel dry method in an amount which is 2-4 times the weight of the extractum; gradient eluting with chloroform-acetone solution, mixing the same parts, collecting eluate, and concentrating;

(3) high-pressure liquid chromatography separation and purification: and further separating and purifying the 7:3 part of the eluent by using high pressure liquid chromatography to obtain the required quinoline alkaloid.

3. The method of claim 2, wherein: in the step (3), the compound separated and purified by the high pressure liquid chromatography is dissolved by pure methanol again, and then the pure methanol is used as a mobile phase to be separated by gel column chromatography for further separation and purification.

4. The method of claim 2, wherein: in the step (3), the high pressure liquid chromatography separation and purification adopts C with the size of 21.2mm multiplied by 250mm and 5 mu m₁₈And (3) carrying out chromatographic column chromatography, wherein the flow rate is 20mL/min, the mobile phase is 52% methanol, the detection wavelength of an ultraviolet detector is 334nm, 200 mu L of sample is injected each time, a chromatographic peak of 30.2min is collected, and the chromatographic peak is evaporated to dryness after multiple accumulation.

5. The method of claim 2, wherein: in the step (2), before the extractum is subjected to silica gel column chromatography and coarse separation, the extractum is dissolved by pure methanol or pure ethanol or pure acetone with the weight ratio of 1.5-3 times, and then the extractum is mixed by 80-100 meshes of silica gel with the weight ratio of 0.8-1.2 times.

6. The method of claim 2, wherein: in the step (2), the gradient elution is carried out, wherein the volume ratio of the chloroform-acetone solution is 1:0, 20:1, 9:1, 8:2, 7:3, 6:4, 1:1 and 1: 2.

7. The application of the quinoline alkaloid of claim 1 or the quinoline alkaloid prepared by the preparation method of any one of claims 2 to 6 in tobacco mosaic virus resistance.